Cortisol, a biomarker of stress, has recently been shown to have potential in evaluating the physiological state of individuals diagnosed with stress-related conditions including chronic fatigue syndrome. Noninvasive techniques to extract biomarkers from the body are a topic of considerable interest. One such technique to achieve this is known as reverse iontophoresis (RI) which is capable of extracting biomolecules through the skin. Unfortunately, however, the extracted levels are often considerably lower in concentration than those found in blood, thereby requiring a very sensitive analytical method with a low limit of detection. A promising sensing approach, which is well suited to handle such samples, is Surface Plasmon Resonance (SPR) spectroscopy. When coupled with aptamer modified surfaces, such sensors can achieve both selectivity and the required sensitivity. In this study, fabrication and characterization of a RIbased SPR biosensor for the measurement of cortisol has been developed. The optical mount and diffusion cell were both fabricated through the use of 3D printing techniques. The SPR sensor was configured to employ a prism couplerbased arrangement with a laser generation module and CCD line sensor. Cortisol-specific DNA aptamers were immobilized onto a gold surface to achieve the necessary selectivity. For demonstration purposes, cortisol was extracted by the RI system using a skin phantom flow system capable of generating time dependent concentration profiles. The captured sample was then transported using a micro-fluidic platform from the RI collection site to the SPR sensor for real-time monitoring. Analysis and system control was accomplished within a developed LabVIEW® program.